Preparation method of magnetically controlled piezoelectric short fiber oriented microsphere stent

Abstract

The application discloses a preparation method of a magnetically controlled piezoelectric short fiber oriented microsphere scaffold, and comprises the following steps: 1) preparing a biomaterial electrospun fiber membrane containing magnetic nanoparticles and piezoelectric particles; 2) grinding the treated fiber membrane into short fibers; 3) dispersing the short fibers in a microsphere preparation solution to prepare magnetically controlled piezoelectric microspheres; 4) dispersing the prepared microspheres in a solution after being treated with a washing liquid to obtain magnetically controlled piezoelectric microspheres wrapped with short fibers; and 5) applying an external magnetic field to the magnetically controlled piezoelectric microspheres to obtain a piezoelectric short fiber oriented microsphere scaffold. The magnetically controlled piezoelectric short fiber oriented microsphere scaffold prepared by the application has good biocompatibility and in-vivo degradability; under the regulation of an external magnetic field, the mechanical force acting on the magnetic nanoparticles in the internal magnetically controlled piezoelectric short fiber oriented microsphere scaffold is applied to the piezoelectric nanoparticles, so that the piezoelectric nanoparticles generate a piezoelectric effect and act on cells and tissues in contact with the piezoelectric nanoparticles, thereby realizing non-invasive and precise regulation of the piezoelectric effect.

Description

Technical Field

[0001] This invention belongs to the field of implantable medical biomaterials in tissue engineering and regenerative medicine, specifically relating to a method for preparing and applying a magnetically controlled piezoelectric short fiber oriented microsphere scaffold for treating peripheral nerve injury. Background Technology

[0002] Peripheral nerve injury is a common clinical condition. The nerve bundle is the basic unit of the peripheral nerve, composed of nerve fibers, Schwann cells, blood vessels, and surrounding connective tissue. Compared to the central nervous system, the peripheral nervous system exhibits a significantly greater regenerative capacity after injury. Schwann cells in the peripheral nervous system are regenerative cells that retain their repair capabilities even after nerve injury. At the time of injury, the distal nerve region undergoes Wallerian degeneration, creating a potential microenvironment for subsequent axonal regeneration. After dedifferentiation, Schwann cells proliferate and extend longitudinally to form Büngner's bands, helping to guide axonal regeneration; they also secrete various cytokines, growth factors, neurotrophic factors, and extracellular matrix molecules, promoting neuronal survival and axonal regeneration. Under the influence of these factors, the injured neuron rapidly activates its intrinsic program and regenerates towards its distal target. Upon contact with the regenerating axon, Schwann cells regenerate the axon and repel infiltrating macrophages, completing the peripheral nerve repair process.

[0003] Severe peripheral nerve injuries rarely heal spontaneously and, without surgical or pharmacological intervention, can lead to permanent loss of nerve function, such as motor dysfunction, chronic pain, and muscle atrophy. Currently, autologous transplantation is the "gold standard" for treating peripheral nerve injuries. However, this method has limitations such as insufficient donor nerves, donor site infection, and difficulty in accommodating long-distance defects, leading to secondary damage and sensory loss at the donor site. Therefore, artificial nerve grafts have become a research hotspot. However, existing nerve grafts do not yet create a microenvironment that fully meets the requirements for peripheral nerve regeneration. Therefore, their regeneration effect is still inferior to autologous nerve grafts and cannot adequately meet clinical needs, necessitating further improvements in graft performance.

[0004] Ideal tissue-engineered neural grafts should be a composite of a bio-scaffold and its internal nutritional factors and biomimetic structures. These biomimetic structures can provide a suitable microenvironment for the migration, proliferation, and functionalization of nerve cells, and provide contact guidance for the directional growth of axons, thereby improving the accuracy of nerve alignment. However, due to the complexity of natural neural structures, fabricating artificial neural grafts with micro / nano-scale nerve-like structures remains a significant challenge. In recent years, various biomimetic microspheres with unique structures and properties have been developed as cell carriers and drug delivery carriers. As injectable scaffolds with excellent signal delivery capabilities, microspheres can be injected into irregular defects or damaged tissues, thus shortening recovery time by several times compared to traditional 3D scaffold surgery. To date, microspheres of different shapes and structures have been used in tissue engineering research.

[0005] Short fibers can effectively mimic the physical microenvironment of neural tissue, providing a suitable extracellular environment for cell growth and thus promoting nerve cell development. Examples include core-bound fibers, nanofibers, hollow fibers, and nanofibers. These fibers can effectively mimic the nanoscale to microscale morphology and structure of fibrous components in the neural emulsion membrane (ECM) and are widely used in tissue regeneration. Homogenized short fibers can effectively mimic ECM-like structures, offering greater potential for tissue repair. Meanwhile, nanofiber microspheres possess a large specific surface area, resulting in high drug loading efficiency. Therefore, combining the advantages of both short fibers and microspheres not only simulates the ECM-like morphology but also allows for injection, enabling the treatment of traumatic diseases. Microspheres have a high surface area to volume ratio, providing sufficient space for cell growth. Furthermore, microspheres with functional structures (drug-loaded, core-shell) are easy to prepare and can effectively enhance the delivery of cells and bioactive molecules. Therefore, functional nanofiber microspheres, as a novel injectable scaffold, have attracted widespread attention in recent years and are expected to play a significant role in the repair of peripheral nerve defects.

[0006] Studies have found that various external physical stimuli, such as electricity, magnetism, light, and sound, can influence biological activities and regulate cellular behavior. Among these, magnetic stimulation exhibits advantages over other types of stimulation. Magnetokinesis has significant advantages in non-invasive treatment, non-contact controllability, and high penetration ability, and has been widely applied in tissue engineering, such as in cell 3D printing and inducing stem cell differentiation. Under low-frequency magnetic fields, the movement of magnetic nanoparticles (MNPs) can induce magnetomechanical actuation of loaded magnetic materials, enabling non-invasive treatment and magnetically controlled drug delivery in the biomedical field. Electrical signals are considered to play an important role in promoting nerve cell function during neurogenesis and are also an important consideration in designing nerve repair scaffolds. Therefore, piezoelectric materials with piezoelectric effects have become a research hotspot in tissue engineering. Piezoelectric biomaterials can generate an electric field (EF) when subjected to stress (deformation). Therefore, they can improve tissue repair and regeneration by stimulating signaling pathways. A series of studies have shown that the potential induced by piezoelectric polymers and ceramics may stimulate cellular responses and bioactivity in hard tissues, showing great potential in non-invasive treatment of nerve regeneration. Piezoelectric scaffolds have been used as an effective material for repairing damaged nerves. Therefore, short fibers with added MNPs (such as Fe3O4, Fe2O3) and piezoelectric particles (such as BaTiO3, ZnO) can be prepared. Under the control of an external magnetic field, the MNPs can apply mechanical force to the piezoelectric particles on the fiber, causing them to produce a piezoelectric effect, thereby stimulating and regulating the surrounding cells and tissues accordingly. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing and applying a magnetically controlled piezoelectric short fiber oriented microsphere scaffold for treating peripheral nerve injuries, thereby achieving a magnetically controlled piezoelectric effect. This addresses the difficulties in achieving non-invasive, in vivo piezoelectric effects in existing technologies, as well as the problem of disordered microsphere distribution hindering the directional growth of cells. The microspheres of this invention respond to an external magnetic field through their own magnetic nanoparticles, which act on the BaTiO3 piezoelectric ceramic particles on the short fibers, causing the microspheres to produce a piezoelectric effect. Under the influence of an external magnetic field, the microspheres and the short fibers within them can be oriented in a specific direction. This anisotropic topology effectively guides Schwann cell elongation and migration, and axonal extension, accelerating the regeneration and repair of peripheral nerves. Compared to traditional microsphere scaffolds, this invention promotes the directional elongation of severed nerves through anisotropic topology and innovatively uses magnetic nanoparticles to respond to a magnetic field and generate mechanical force acting on the piezoelectric particles on the same short fiber, thus producing a magnetically controlled piezoelectric effect. This avoids the problem of traditional piezoelectric materials failing to generate a piezoelectric effect after surgery when pressure cannot be applied normally. The magnetron piezoelectric method is simple and easy to implement. It only requires placing a magnet near the magnetron piezoelectric microspheres, causing the microspheres to be subjected to an external magnetic field and generate a certain mechanical force that acts on the piezoelectric nanoparticles, thus producing a piezoelectric phenomenon. Therefore, this invention has significant application value in addressing the gap in the market for related products.

[0008] To address the aforementioned problems, the present invention adopts the following technical solution:

[0009] A method for fabricating a magnetron-controlled piezoelectric short fiber oriented microsphere scaffold includes the following steps:

[0010] (1) Surface modification was used to modify the surface of magnetic nanoparticles and piezoelectric nanoparticles to improve particle dispersibility, degradability and biocompatibility;

[0011] (2) Biomaterial-based micro / nano long fibers with a diameter of nanometers and loaded with magnetic nanoparticles and piezoelectric nanoparticles were prepared by nanofiber preparation technology.

[0012] (3) Disperse long fibers uniformly in a solution and use micro-nano processing technology to prepare short fibers with a length distribution within a certain range;

[0013] (4) Disperse a certain mass of short fibers in the microsphere preparation solution;

[0014] (5) Short fiber solution was prepared into magnetron piezoelectric short fiber microspheres using microsphere preparation technology, and the microspheres were separated from the liquid phase by cross-linking method;

[0015] (6) The formed microspheres are filtered through a sieve, washed and dispersed in a solution to obtain a magnetron piezoelectric short fiber microsphere scaffold.

[0016] Preferably, the diameter of the biomaterial-based micro / nano fibers is 10 nm-500 μm.

[0017] Preferably, the tissue-engineered graft in this invention is one or more of the spinnable materials such as ovalbumin (OVA), polycaprolactone (PCL), silk fibroin (SF), and chitosan (CS).

[0018] Preferably, the concentration of piezoelectric nanoparticles in step (1) is 0-2 g / mL, and the concentration that produces the maximum piezoelectric effect is selected for subsequent operations.

[0019] Preferably, the concentration of magnetic nanoparticles in step (1) is 0-2 g / mL. The concentration that can generate the best piezoelectric effect by responding to the magnetic field is selected for subsequent operations.

[0020] Preferably, the nanofiber preparation technology in step (2) involves the following processes: preparation of biomaterial solution, ultrasonic vibration to uniformly disperse magnetic nanoparticles and piezoelectric nanoparticles in biomaterial solution, nanofiber preparation process, and removal of organic solvent.

[0021] Preferably, the concentration of short fibers in step (3) is 0.1-1 g / mL; the preparation time of short fibers is 1-20 min; and the length of short fibers that can produce microspheres of a suitable size is selected.

[0022] Preferably, the magnetic nanoparticles in step (1) are one or more of Fe, Co, Ni monomers and their alloy nanoparticles, and metal oxide nanoparticles; the piezoelectric nanoparticles are ZnO, perovskite (SrTiO3, BaTiO3, Bi 0.5 Na 0.5 The nanoparticles are one or more of TiO3, KNbO3 or ZnSnO3, and BiOX (where X = Cl, Br, I, etc.); the concentration range of the nanoparticles is 0.1-200 mg / mL.

[0023] Preferably, the surface modification method in step (1) is one or more of the following: inorganic metal modification (Au, Ag, etc.), organic small molecule modification (oleic acid, tannic acid, sodium oleate, dodecylamine, γ-cyclodextrin, etc.), polymer modification (polydopamine, chitosan, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, polyacrylic acid, polyethyleneimine, etc.), and silanization modification; the dopamine modification concentration is 2 mg / mL, and the chitosan modification concentration is 10-20 mg / mL.

[0024] Preferably, the nanofiber preparation method in step (2) is one or more of electrospinning technology, microfluidics technology, 3D printing technology, and near-field direct writing technology; wherein, the electrospinning parameters are: flow rate of 5-25μL / min, voltage of 10-25kV, receiving distance of 10-20cm, receiving method of flat plate receiving or roller collection, roller receiving speed of 1000-2000rpm, and spinning time of 6-12h; the microfluidics technology parameters are: propulsion speed of 1-300μL / min; the 3D printing technology parameters are: print head temperature of 5-15℃, printing platform temperature of -10℃, and printing speed of 5-20mm / s; the near-field direct writing parameters are: needle inner diameter of 0.5mm, melt thrust of 0.2MPa, fiber spacing of each sample of 1.0mm, receiving distance of 3-6cm, receiving plate moving speed of 3-7mm / s, and melt temperature of 100-150℃;

[0025] Preferably, the biomaterials mentioned in step (2) are one or more of the following: natural biomaterials (collagen, fibroin, gelatin, silk fibroin, egg white protein, chitosan, cellulose, hyaluronic acid, alginate, etc.) and synthetic biomaterials (polycaprolactone, polyvinylpyrrolidone, polyethylene glycol, polylactic acid, etc.);

[0026] Preferably, the long fiber dispersion solution in step (3) is either PBS or physiological saline; the short fiber preparation method is one or more of the following: grinding, homogenization, micro-shearing, liquid nitrogen brittle fracture, freeze drying and then pulverization; the short fiber length distribution range is 5-150 μm, the fiber diameter distribution range is 20-500 nm, the length distribution range is determined according to the time of the short fiber preparation method, and the diameter distribution range is determined according to factors such as the flow rate, rotation speed, and voltage of the fiber preparation method;

[0027] Preferably, the microsphere preparation solution in step (4) is one or more of N,N-dimethylformamide (DMF), hexafluoroisopropanol (HFIP), formic acid, chloroform (CHCl3), acetic acid, gellan gum solution, sodium alginate solution, and oils containing surfactants; in the microsphere collection method, the collection solution is Ca 2+ The solution contains one or more of the following: formaldehyde, glutaraldehyde, etc.; the concentration of the gellan gum solution is 0.1-1% (w / v), the concentration of the sodium alginate solution is 0.5-5 g / mL, and the concentration of Ca... 2+ The concentration is 0.1-1M, and the surfactant is one of Span or Tween;

[0028] Preferably, the microsphere preparation technology in step (5) is one of the following: spray drying, electrospraying, water-in-oil emulsification, microfluidics, freeze drying, emulsion-solvent evaporation, phase separation, and reverse suspension crosslinking; the crosslinking method is ionic crosslinking (Ca... 2+ The magnetically controlled piezoelectric microspheres are one of cross-linking, photocross-linking, and thermo-cross-linking; the diameter of the magnetically controlled piezoelectric microspheres is distributed between 10 nm and 500 μm, and magnetically controlled piezoelectric microspheres of different diameters can be prepared according to actual needs.

[0029] Preferably, the washing liquid used in the microsphere washing method in step (6) is one or more of anhydrous ethanol, 75% ethanol, isopropanol, acetone, etc.; the external magnetic field driving source of the magnetically controlled piezoelectric microsphere is a static magnetic field or alternating magnetic field with magnetic field strength ranging from low to high intensity (10mT-2T).

[0030] The beneficial effects of this invention are:

[0031] (1) The present invention prepares electrospun fibers into short fibers, which can better simulate ECM-like structures, which is conducive to the three-dimensional growth of cells and avoids problems such as cell non-adhesion and difficulty in proliferation caused by different physical environments, and is closer to the real microenvironment for cell growth.

[0032] (2) The present invention prepares short fibers into microspheres, which can realize the in vivo delivery of ECM-like structures;

[0033] (3) The present invention adds magnetic nanoparticles and piezoelectric particles to microspheres, which can realize remote control of the movement of microspheres and achieve non-invasive magnetoelectric conversion under the control of external magnetic field, promote nerve damage repair, and thus realize personalized non-invasive nerve damage repair.

[0034] (4) The magnetically controlled piezoelectric short fiber oriented microspheres of the present invention can be encapsulated in nerve guiding conduits to promote the repair of peripheral nerve defects. They can also be used as injectable magnetic piezoelectric functional scaffolds for the repair of wounds in various tissues and organs.

[0035] (5) The magnetically controlled piezoelectric short fiber oriented microsphere scaffold for tissue repair prepared by the present invention has good biocompatibility, can be well loaded with magnetic nanoparticles and piezoelectric nanoparticles, and can be used for remote wireless magnetic control to generate piezoelectric effect for various tissue repairs.

[0036] (6) The preparation process of this invention is simple, the materials are readily available, and the cost is low. It can be used as an ideal biological functional material and has good potential applications in the field of peripheral nerve regeneration. This invention can be extended to the fields of spinal cord nerve repair, skin repair, and bone tissue repair, and has good potential application value. Attached Figure Description

[0037] Figure 1 One method for surface modification of magnetic and piezoelectric nanoparticles: a schematic diagram of dopamine modification;

[0038] Figure 2 SEM images of the modified magnetic nanoparticles;

[0039] Figure 3 TEM image of the modified piezoelectric particles;

[0040] Figure 4 SEM image of long nanofibers;

[0041] Figure 5 SEM image of short nanofibers;

[0042] Figure 6 Optical mirror image of a magnetron-controlled piezoelectric microsphere;

[0043] Figure 7 SEM images of magnetron-controlled piezoelectric microspheres;

[0044] Figure 8 Fluorescence images of magnetron-controlled piezoelectric microspheres co-cultured with Schwann cells;

[0045] Figure 9 This is a flowchart of the preparation method of the present invention. Detailed Implementation

[0046] The present invention will be further illustrated below with reference to examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0047] Example 1

[0048] A magneto-controlled piezoelectric short fiber oriented microsphere scaffold for treating peripheral nerve injury is specifically prepared by the following steps:

[0049] (1) Dissolve 1.5g of OVA powder and 0.2g of PEO in 10mL of a mixed solution of hexafluoroisopropanol (HFIP) / formic acid (3:1), add 20mg of Fe3O4 and ZnO particles respectively, and mix thoroughly by ultrasonic vibration;

[0050] (2) The OVA solution was prepared into an electrospun fiber membrane by electrospinning technology. The electrospinning parameters were: flow rate 10 μL / min, voltage 20 kV, receiving distance d = 15 cm, roller collection, and rotation speed 1500 rpm.

[0051] (3) Place 1g of electrospun fiber in a 1.5mL centrifuge tube and grind the electrospun membrane treated with anhydrous ethanol into short fibers using a homogenizer for 10min; dissolve 0.6g of gellan gum in 100mL of deionized water to prepare a 0.6% w / v gellan gum solution; disperse the ground 1g of short fibers into 10mL of gellan gum solution;

[0052] (4) Microspheres were prepared using a dual-channel micro-injection pump and microfluidic technology. The dispersed phase consisted of OVA short fibers and gellan gum solution, and the mobile phase consisted of a paraffin oil solution containing 5% wt Span80. The receiving device consisted of an equal volume mixture of 1M CaCl2 solution and paraffin oil. The flow rate ratio of the microfluidic system was 1:1000.

[0053] (5) The prepared microspheres were filtered through a sieve and treated with hot ethanol, and then dispersed in 5 mL of PBS solution to obtain magnetron piezoelectric microspheres;

[0054] (6) When co-cultured with cells or applied in vivo, a magnetic field can be applied to the outside to remotely control the microspheres, so that the microspheres respond to the magnetic field to generate mechanical force, which acts on the piezoelectric material to generate the piezoelectric effect and form an array of oriented microspheres to guide the directional growth of new cells.

[0055] Example 2

[0056] A magneto-controlled piezoelectric short fiber oriented microsphere scaffold for treating peripheral nerve injury is specifically prepared by the following steps:

[0057] (1) Dissolve 1g of PCL particles in 10mL of hexafluoroisopropanol (HFIP) solution, add 0.2g of Fe3O4 and BaTiO3 particles respectively, and mix thoroughly by ultrasonic vibration;

[0058] (2) PCL solution was processed into fibers using 3D printing technology: The PCL solution was loaded into a 5mL syringe and extruded through a needle with an inner diameter of 0.26mm at a printing speed of 15mm / s. A low-temperature printing platform was used to rapidly solidify the extruded filaments and maintain the solidification of the printing support. The temperature of the print head was set to 8℃, and the temperature of the printing platform was set to -10℃.

[0059] (3) Place 1g of PCL fiber in a 1.5mL centrifuge tube, place the centrifuge tube in liquid nitrogen for 30min, and use the liquid nitrogen brittle fracture method to grind the fiber treated with anhydrous ethanol into short fibers for 10min.

[0060] (4) Microspheres were prepared using a dual-channel micro-injection pump and microfluidic technology. The dispersed phase consisted of PCL short fibers and sodium alginate solution, and the mobile phase consisted of a decanol solution containing 5% Span 80. The receiving device was a solution prepared by mixing equal volumes of 1M CaCl2 and decanol. The flow rate ratio of the microfluidic system was 1:1000.

[0061] (5) The microspheres were filtered through a sieve and treated with hot ethanol, and then dispersed in 5 mL of PBS solution to obtain magnetron piezoelectric microspheres;

[0062] (6) When co-cultured with cells or applied in vivo, a magnetic field can be applied to the outside to remotely control the microspheres, so that the microspheres respond to the magnetic field to generate mechanical force, which acts on the piezoelectric material to generate the piezoelectric effect and form an array of oriented microspheres to guide the directional growth of new cells.

[0063] Example 3

[0064] A magneto-controlled piezoelectric short fiber oriented microsphere scaffold for treating peripheral nerve injury is specifically prepared by the following steps:

[0065] (1) Dissolve 3g of chitosan (CS) powder and 0.2g of sodium alginate in 100mL of acetic acid (2%) to prepare a 3% chitosan solution. Add 20mg of Fe3O4 and BaTiO3 particles to 10mL of chitosan solution and mix thoroughly by ultrasonic vibration.

[0066] (2) The CS solution was prepared into an electrospun fiber membrane by electrospinning technology. The electrospinning parameters were: flow rate 15 μL / min, voltage 20 kV, receiving distance d = 15 cm, roller collection, and rotation speed 2000 rpm.

[0067] (3) Place 1g of electrospun chitosan fiber in a 1.5mL centrifuge tube and grind the electrospun membrane treated with anhydrous ethanol into short fibers using a grinder for 10min.

[0068] (4) Microspheres were prepared by electrospraying. The fiber solution was sprayed into the metal plate through a nozzle to form microsphere particles. An electrostatic potential of 8kV was applied to the electrospraying device to generate droplets. The droplets were collected in a 10% CaCl2 solution and rapidly crosslinked to form microspheres.

[0069] (5) The microspheres were filtered through a sieve and treated with hot ethanol, and then dispersed in 5 mL of PBS solution to obtain magnetron piezoelectric microspheres;

[0070] (6) When co-cultured with cells or applied in vivo, a magnetic field can be applied to the outside to remotely control the microspheres, so that the microspheres respond to the magnetic field to generate mechanical force, which acts on the piezoelectric material to generate the piezoelectric effect and form an array of oriented microspheres to guide the directional growth of new cells.

[0071] Example 4

[0072] A magneto-controlled piezoelectric short fiber oriented microsphere scaffold for treating peripheral nerve injury is specifically prepared by the following steps:

[0073] (1) Dissolve 1.5g OVA and 0.2g PEO in a hexafluoroisopropanol (HFIP) / formic acid (3:1) solution, add 10mg of Fe2O3 and ZnO particles respectively, and mix thoroughly by ultrasonic vibration;

[0074] (2) The OVA solution was prepared into an electrospun fiber membrane using 3D printing technology: The OVA solution was loaded into a 5mL syringe and extruded through a needle with an inner diameter of 0.26mm at a printing speed of 20mm / s. A low-temperature printing platform was used to rapidly solidify the extruded filaments and maintain the solidification of the printing support. The temperature of the print head was set to 8℃, and the temperature of the printing platform was set to -10℃.

[0075] (3) Place 1g of OVA fiber in a 1.5mL centrifuge tube and use a homogenizer to grind the electrospun membrane treated with anhydrous ethanol into short fibers for 10min.

[0076] (4) Microspheres were prepared using the O / W emulsification-solvent evaporation method. 1 g of the prepared short fibers were dispersed in 10 mL of an aqueous phase containing sodium alginate. The oil phase was slowly added, and the liquid was stirred at 2000 rpm. Ca... 2+ Ion cross-linking extraction microspheres;

[0077] (5) The microspheres were filtered through a sieve and treated with hot ethanol, and then dispersed in 5 mL of PBS solution to obtain magnetron piezoelectric microspheres;

[0078] (6) When co-cultured with cells or applied in vivo, a magnetic field can be applied to the outside to remotely control the microspheres, so that the microspheres respond to the magnetic field to generate mechanical force, which acts on the piezoelectric material to generate the piezoelectric effect and form an array of oriented microspheres to guide the directional growth of new cells.

[0079] Example 5

[0080] A magneto-controlled piezoelectric short fiber oriented microsphere scaffold for treating peripheral nerve injury is specifically prepared by the following steps:

[0081] (1) Dissolve 0.3g CS in 2% acetic acid solution to make 10mL 3% CS solution, add 20mg Fe3O4 and 10mg ZnO particles respectively, and mix thoroughly by ultrasonic vibration.

[0082] (2) The CS solution was prepared into an electrospun fiber membrane by electrospinning technology. The electrospinning parameters were: flow rate 25 μL / min, voltage 20 kV, receiving distance d = 15 cm, and plate collection was used.

[0083] (3) Place 1g of electrospun CS fiber in a 1.5mL centrifuge tube and use a homogenizer to grind the electrospun membrane treated with anhydrous ethanol into short fibers for 10min.

[0084] (4) Microspheres were prepared by electrostatic spraying using a microsphere collector, wherein the dispersed phase was CS short fiber and gellan gum solution, the mobile phase was a glycerol solution containing 5% Span 80; the receiving device was a solution prepared by mixing equal volumes of 1M CaCl2 and glycerol; the flow rate ratio of the microfluidic system was 1:1000.

[0085] (5) The microspheres were filtered through a sieve and treated with hot ethanol, and then dispersed in 5 mL of PBS solution to obtain magnetron piezoelectric microspheres;

[0086] (6) When co-cultured with cells or applied in vivo, a magnetic field can be applied to the outside to remotely control the microspheres, so that the microspheres respond to the magnetic field to generate mechanical force, which acts on the piezoelectric material to generate the piezoelectric effect and form an array of oriented microspheres to guide the directional growth of new cells.

[0087] Example 6

[0088] A magneto-controlled piezoelectric short fiber oriented microsphere scaffold for treating peripheral nerve injury is specifically prepared by the following steps:

[0089] (1) Dissolve 15% silk fibroin (SF) and 2% PEO in a hexafluoroisopropanol (HFIP) / formic acid (3:1) solution, add 20 mg of Fe3O4 and BaTiO3 particles respectively, and mix thoroughly by ultrasonic vibration;

[0090] (2) The OVA solution was prepared into an electrospun fiber membrane using 3D printing technology: The silk fibroin solution was loaded into a 5mL syringe and extruded through a needle with an inner diameter of 0.26mm at a printing speed of 18mm / s. A low-temperature printing platform was used to rapidly solidify the extruded filaments and maintain the solidification of the printing support. The temperature of the print head was set to 8℃, and the temperature of the printing platform was set to -10℃.

[0091] (3) Place 1g of silk fibroin fiber in a 1.5mL centrifuge tube, place the centrifuge tube in liquid nitrogen for 30min, and use the liquid nitrogen brittle fracture method to grind the electrospun membrane treated with anhydrous ethanol into short fibers for 10min.

[0092] (4) Microspheres were prepared using a dual-channel micro-injection pump and microfluidic technology. The dispersed phase consisted of short silk fibroin fibers and gellan gum solution, and the mobile phase consisted of a paraffin oil solution containing 5% Span 80. The receiving device was a solution prepared by mixing equal volumes of 1M CaCl2 and paraffin oil. The flow rate ratio of the microfluidic system was 1:1000.

[0093] (5) The microspheres were filtered through a sieve and treated with hot ethanol, and then dispersed in 5 mL of PBS solution to obtain magnetron piezoelectric microspheres;

[0094] (6) When co-cultured with cells or applied in vivo, a magnetic field can be applied to the outside to remotely control the microspheres, so that the microspheres respond to the magnetic field to generate mechanical force, which acts on the piezoelectric material to generate the piezoelectric effect and form an array of oriented microspheres to guide the directional growth of new cells.

[0095] Example 7

[0096] A magneto-controlled piezoelectric short fiber oriented microsphere scaffold for treating peripheral nerve injury is specifically prepared by the following steps:

[0097] (1) Dissolve 10g PCL and 5g silk fibroin (SF) in hexafluoroisopropanol (HFIP) solution, add 10mg CoFe2O4 and 20mg BaTiO3 particles respectively, and mix thoroughly by ultrasonic vibration.

[0098] (2) The PCL / SF solution was prepared into an electrospun fiber membrane using near-field direct writing technology; the inner diameter of the needle was 0.5 mm, the melt thrust was 0.2 MPa, the fiber spacing of each sample was set to 1.0 mm, the receiving distance was 4 cm, and the receiving plate moving speed was 5 mm / s.

[0099] (3) Place 1g of PCL / SF fiber in a 1.5mL centrifuge tube, freeze dry the electrospun membrane treated with anhydrous ethanol using freeze-drying technology, and then grind it into short fibers.

[0100] (4) Microspheres were prepared using a dual-channel microinjection pump and microfluidic technology. The dispersed phase was PCL / SF short fibers and alginate solution, and the mobile phase was a glycerol solution containing 15% Span 80. The receiving device was a solution prepared by mixing equal volumes of 1M CaCl2 and glycerol. The flow rate ratio of the microfluidic system was 1:500.

[0101] (5) The microspheres were filtered through a sieve and treated with hot ethanol, and then dispersed in 5 mL of PBS solution to obtain magnetron piezoelectric microspheres;

[0102] (6) When co-cultured with cells or applied in vivo, a magnetic field can be applied to the outside to remotely control the microspheres, so that the microspheres respond to the magnetic field to generate mechanical force, which acts on the piezoelectric material to generate the piezoelectric effect and form an array of oriented microspheres to guide the directional growth of new cells.

[0103] Example 8

[0104] A magneto-controlled piezoelectric short fiber oriented microsphere scaffold for treating peripheral nerve injury is specifically prepared by the following steps:

[0105] (1) Dissolve 1g PCL and 1g PVDF in 10mL hexafluoroisopropanol solution, add 10mg Co3O4 particles, and mix thoroughly by ultrasonic vibration.

[0106] (2) The PCL solution was prepared into an electrospun fiber membrane by electrospinning technology. The electrospinning parameters were: flow rate 10 μL / min, voltage 20 kV, receiving distance d = 15 cm, roller collection, and rotation speed 1000 rpm.

[0107] (3) Place 1g of electrospun fiber in a 1.5mL centrifuge tube and use a homogenizer to grind the electrospun membrane treated with anhydrous ethanol into short fibers for 10min.

[0108] (4) Microspheres were prepared using a dual-channel micro-injection pump and microfluidic technology. The dispersed phase consisted of PCL short fibers and sodium alginate solution, and the mobile phase consisted of sunflower seed oil solution containing 5% Span 80. The receiving device was a solution prepared by mixing equal volumes of 1M CaCl2 and sunflower seed oil. The flow rate ratio of the microfluidic system was 1:1500.

[0109] (5) The microspheres were filtered through a sieve and treated with hot ethanol, and then dispersed in 5 mL of PBS solution to obtain magnetron piezoelectric microspheres;

[0110] (6) When co-cultured with cells or applied in vivo, a magnetic field can be applied to the outside to remotely control the microspheres, so that the microspheres respond to the magnetic field to generate mechanical force, which acts on the piezoelectric material to generate the piezoelectric effect and form an array of oriented microspheres to guide the directional growth of new cells.

Claims

1. A method for fabricating a magnetron-controlled piezoelectric short fiber oriented microsphere scaffold, characterized in that, Includes the following steps: (1) Surface modification is used to modify the surface of magnetic nanoparticles and piezoelectric nanoparticles to improve particle dispersibility, degradability and biocompatibility; the magnetic nanoparticles are one or more of Fe, Co, Ni monomers and their alloy nanoparticles and metal oxide nanoparticles; the piezoelectric nanoparticles are one or more of ZnO, perovskite, BiOX, X=Cl, Br, I; (2) Biomaterial-based micro / nano long fibers with diameters in the nanometer range, loaded with magnetic nanoparticles and piezoelectric nanoparticles, were prepared using nanofiber preparation technology. (3) Disperse the long fibers uniformly in the solution and use micro-nano processing technology to prepare short fibers with a length distribution within a certain range; (4) Disperse the short fibers in the microsphere preparation solution; (5) The short fiber solution was prepared into magnetron piezoelectric short fiber microspheres using microsphere preparation technology, and the microspheres were separated from the liquid phase by cross-linking method; (6) The formed microspheres are filtered through a sieve, washed and dispersed in a solution to obtain a magnetron piezoelectric short fiber microsphere scaffold.

2. The method for preparing a magnetron-controlled piezoelectric short fiber oriented microsphere scaffold according to claim 1, characterized in that, The diameter of the biomaterial-based micro / nano fibers is 10 nm-500 µm.

3. The method for preparing a magnetron-controlled piezoelectric short fiber oriented microsphere scaffold according to claim 1, characterized in that, The concentration range of the magnetic nanoparticles and piezoelectric nanoparticles is 0.1-200 mg / mL.

4. The method for preparing a magnetron-controlled piezoelectric short fiber oriented microsphere scaffold according to claim 1, characterized in that, The surface modification method in step (1) is one or more of the following: inorganic metal modification, organic small molecule modification, polymer modification, and silanization modification.

5. The method for preparing a magnetron-controlled piezoelectric short fiber oriented microsphere scaffold according to claim 1, characterized in that, The nanofiber preparation technology mentioned in step (2) is one or more of electrospinning, microfluidics, 3D printing, and near-field direct writing. The electrospinning parameters are: flow rate of 5-25 µL / min, voltage of 10-25 kV, receiving distance of 10-20 cm, receiving method of flat plate receiving or roller collection, roller receiving speed of 1000-2000 rpm, and spinning time of 6-12 h. The microfluidics parameters are: propulsion speed of 1-300 µL / min. The 3D printing parameters are: print head temperature of 5-15°C, printing platform temperature of −10°C, and printing speed of 5-20 mm / s. The near-field direct writing parameters are: needle inner diameter of 0.5 mm, melt thrust of 0.2 MPa, fiber spacing of 1.0 mm per sample, receiving distance of 3-6 cm, receiving plate moving speed of 3-7 mm / s, and melt temperature of 100-150 °C. ℃.

6. The method for preparing a magnetron-controlled piezoelectric short fiber oriented microsphere scaffold according to claim 1, characterized in that, The biomaterials mentioned in step (2) are one or more of natural biomaterials and synthetic biomaterials.

7. The method for preparing a magnetron-controlled piezoelectric short fiber oriented microsphere scaffold according to claim 1, characterized in that, The long fiber dispersion solution in step (3) is either PBS or physiological saline; the short fiber preparation method is one or more of grinding, homogenization, micro-shearing, liquid nitrogen brittle fracture, freeze drying and then pulverization; the short fiber length distribution range is 5-150µm, the fiber diameter distribution range is 20-500nm, the length distribution range is determined according to the time of the short fiber preparation method, and the diameter distribution range is determined according to the flow rate, rotation speed and voltage of the fiber preparation method.

8. The method for preparing a magnetron-controlled piezoelectric short fiber oriented microsphere scaffold according to claim 1, characterized in that, In step (4), the microsphere preparation solution is one or more of N,N-dimethylformamide, hexafluoroisopropanol, formic acid, chloroform, acetic acid, gellan gum solution, sodium alginate solution, and oil containing surfactant; the concentration of gellan gum solution is 0.1-1% w / v, the concentration of sodium alginate solution is 0.5-5 g / mL, and the surfactant is one of Span and Tween.

9. The method for preparing a magnetron-controlled piezoelectric short fiber oriented microsphere scaffold according to claim 1, characterized in that, The microsphere preparation technology mentioned in step (5) is one of spray drying, electrospraying, water-in-oil emulsification, microfluidics, freeze drying, emulsion-solvent evaporation, phase separation, and reverse suspension crosslinking; the crosslinking method is one of ionic crosslinking, photocrosslinking, and thermosensitive crosslinking; the diameter of the magnetron piezoelectric short fiber microspheres is distributed in the range of 10 nm to 500 µm.

10. The method for preparing a magnetron-controlled piezoelectric short fiber oriented microsphere scaffold according to claim 1, characterized in that, In step (6), the microsphere washing method uses one or more of the following washing solutions: anhydrous ethanol, 75% ethanol, isopropanol, and acetone.

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